The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Previous work has demonstrated that translocation occurs through a protein-conducting channel that is formed from a heterotrimeric membrane protein complex (called Sec61p complex in eukaryotes, SecYEG complex in bacteria, and SecYEbeta complex in archaebacteria). Depending on the interacting partner, the channel can function in four different translocation pathways: 1. Co-translational translocation, 2. Post-translational translocation in eukaryotes, 3. Post-translational translocation in bacteria, and 4. Retrotranslocation from the endoplasmic reticulum (ER) into the cytosol. This proposal addresses key aspects of all four translocation modes, with particular emphasis on the least understood retro-translocation pathway. Specifically, we will address the following questions: 1. How is the protein-conducting channel assembled and gated? The planned experiments will clarify how the ribosome binds to the channel in co-translational translocation, how the channel is assembled from several copies of the heterotrimeric membrane protein complex, and how it is opened for translocation. We will also test whether the ER membrane is permeable for small molecules. 2. How does SecA move polypeptides through the channel? We will address the mechanism of posttranslational translocation in bacteria. Specifically, we will test the hypothesis that SecA functions analogously to monomerie helicases to push polypeptides through the channel. 3. How is retro-translocation initiated? We will investigate whether the unfolding of cholera toxin is coupled to its subsequent retro-translocation through the ER membrane. This will involve experiments that address the role of protein disulfide isomerase (PDI) and its oxidase Erol in retro-translocation. 4. How are proteins moved from the ER membrane into the cytosol? The goal of these experiments is to understand how the HMC class I heavy chain is moved into the cytosol during retro-translocation induced by the human cytomegalovirus protein US 11. The planned experiments will identify the components required for poly-ubiquitination of the heavy chain and will test the role of the AAA ATPase p97 and of its partner proteins in extracting proteins from the ER membrane.